Regulated expression of recombinant proteins using RNA viruses

ABSTRACT

The present invention describes cells and constructs for a regulated viral (e.g. alphavirus) expression system, where gene expression is controlled by controlling expression of replicases or nonstructural proteins and/or controlling the amount of such proteins introduced in a cell, which in turn regulates RNA replication and subsequently gene expression. Particularly, this system takes advantage of the high level expression of the alphavirus systems for recombinant protein production and allows for large scale applications without biosafety concerns.

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/094,476, filed on Jul. 29, 1998, the contents ofwhich are entirely incorporated herein by reference.

BACKGROUND AND SUMMARY OF THE INVENTION

[0002] Alphaviruses are single-stranded RNA viruses with a spike proteinenvelope structure. Even though infection usually occurs via areceptor-mediated endocytosis, naked viral RNA can initiate infectionwhen introduced into the cytoplasm of a wide variety of host cells,including vertebrate and invertebrate cells. The 5′ two-thirds of the12-kb viral RNA encodes the four viral nonstructural, or replicaseproteins nsp 1, nsp 2, nsp 3 and nsp 4, required for RNA amplificationin the infected cell. The remaining third of the viral RNA codes for thestructural proteins, the viral capsid and spike proteins, which aretranslated from the subgenomic 26S RNA with its own 26S promoter. Oncethe positive-sense RNA genome of the alphavirus is in the cytoplasm, itserves as the template for synthesis of a complementary negative strandby the virus-encoded replicase. The negative strand serves as thetemplate for additional genomic RNA. Viral RNA replication is extremelyrapid resulting in packaging of high titer alphavirus stocks of up to10¹⁰ units per milliliter.

[0003] Due to its high replication efficiency, the simplicity of thereplication cycle, i.e. the ability of the virus to replicate without aDNA intermediate, and its wide host range, alphaviruses have beenstudied for their potential as virus-based expression vectors. Since thevirus requires only the presence of the replicase to replicate, theregion encoding the replicase and all sequences required in cis forreplication and packaging are maintained and the region encoding thestructural proteins can be replaced with a desired gene. This type ofviral vector with a replication capability is termed a replicon. It iscurrently possible with the alphavirus expression systems to split thereplicon into two distinct replicons (Liljestrom and Garoff (1991)Bio/Technology 9:1356-1361; Xiong el. al. (1989) Science 243:1188-1191).One replicon contains all the viral structural genes and a replicationsignal, and is known as a helper plasmid or RNA. The second repliconcontains the nonstructural proteins coding sequences, a signal forpackaging of the RNA replicon, an expression cassette for expression offoreign genes (under control of the 26S promoter), and is known as theexpression replicon. When the two RNA replicons are transfected intocells, it is possible for the nonstructural proteins of the expressionvector to replicate the helper RNA and express the structural proteins,thus allowing for specific packaging of the expression replicon. Theseviral particles can then be used to infect cells and express protein.The replication of these RNA replicons is very rapid and a single RNAmolecule can replicate up to 100,000 copies or more in 4-6 hours(Wengler, G. (1980) In: The Togaviruses. R. W. Schlesinger (ed.),Academic Press, New York, pp. 459-472).

[0004] The alphavirus expression systems using Semliki forest virus(SFV) (Liljestrom and Garoff (1991) Bio/Technology 9:1356-1361) andSindbis (Xiong et al.(1989) Science 243:1188-1191; Dubensky et al.(1996)J. Virol. 70:508-519) have been extensively used to express recombinantproteins transiently, either as a transient transfection system, or byusing viral particles to infect cells (Lundstrom K. (1997) Cur. Opin.Biolechnol. 8:578-582; Ciccarone et al.(1994) Focus 15: 103-105;Liljestrom, P. (1994) Curr. Opin. Biotechnol. 5:495-500). These systemshave several advantages over other expression systems in that: 1)expression levels are high, and 2) viral particles can be generated andused to infect a variety of cell types so that authentic,post-translationally modified proteins can be made (Lundstrom et al.(1997) Eur. J. Pharmacol. 337:73-81).

[0005] Due to the fact that SFV and Sindbis are human pathogens, thereare disadvantages with the viral expression system because of biosafetyissues and problems with scale up. Under current regulations, the use of>10⁹ viral particles requires a higher level of containment than aBiosafety Level 2 (BL2) laboratory making large scale viral infectionand production, and subsequently, large scale manufacturing of proteins,problematic. Additionally, the viral life cycle is lethal in that aviral infection will kill cells at 48-96 hours post-infection ortransfection, regardless of whether the viral RNA is introduced aspackaged viral particles or by transfection of RNA followed byreplication and protein expression. Some approaches have been describedto control the replication rate of alphavirus RNA such as theoverexpression of the anti-apoptotic protooncogene bcl-2 (Scallan, M. F.et al.(1997) J. Virol. 71:1583-1590), but this strategy only temporarilyslowed viral replication and host cell apoptosis.

[0006] Therefore, there is a need for a non-viral or viral basedexpression system in which one could control the replication rate ofthese replicons so that they are not lethal to the host cell and whichwould allow for large-scale applications. In this system, an alphavirusDNA vector is utilized.

[0007] Alphavirus DNA vectors have been developed for both SFV (BerglundP., Tubulekas I., Liljestrom P., Alphavirus as vectors for genedelivery, Trends Biotechnol 1996, 14: 130-134) and Sinbis (Johanning F.W., Conry R. M., LoBuglio A. F., Wright M., Sumerel L. A., Pike M. J.,Curiel D. T., A Sinbis Virus mRNA polynucleotide vector achievesprolonged and high level heterologous gene expression in vivo, NucleicAcids Res 1995, 23:1495-1501; Herweijer H., Latendresse J. S., WilliamsP., Zhang G., Danko I., Schlesinger S., Wolf J. A., A plasmid-basedself-amplifying Sindbis virus vector, Hum Gene Ther. 1995, 6:1161-1167). In these DNA-RNA layered vectors a eukaryotic promoter isintroduced upstream of the alphavirus replicase genes. DNA istranscribed to RNA from a recombinant eukaryotic promoter in the nucleusand transported to the cytoplasm, where the viral replicase takes overin the same way as during normal replication of alphavirus RNAmolecules. The levels of reporter gene expression is 10-200-fold higherin mouse muscle cells for alphavirus DNA vectors than with conventionalDNA vectors (Johanning, et al., infra, Herweijer, et al., infra;Dubensky, T. W., Driver, D. A., Polo, J. M., Belli, B. A., Latham, E.M., Ibanez, C. E., Chada S., Brumm D., Banks T. A., Mento S. J., et al.,Sinbis virus DNA-based expression vectors: utility for in vitro and invivo gene transfer, J. Virol. 1996, 70: 508-519). Expression istransient in nature as seen in mouse quadriceps, where no expression isdetected 16 days post-injection. Therefore, alphaviruses can beefficiently used as tools in gene therapy for safe short-term geneexpression.

[0008] The present invention relates to RNA virus expression andparticularly to an alphavirus expression system in which one can controlviral RNA replication so that it is not lethal or not substantiallylethal to the host cell, and is therefore amenable to large scaleproduction of RNA and protein. However, any viral expression systemwhich employs RNA self-replication as a mechanism for viralamplification and expression can be used according to the presentinvention, including alphaviruses (animal viruses) such as togaviruses,i.e. Sindbis, SFV, Eastern Equine Encephalitis Virus (EEEV) andVenezuelan Equine Encephalitis Virus (VEEV), and flaviviruses, i.e.yellow fever virus, tick borne encephalitis virus; as well as plantviruses such as tobamoviruses (tobacco mosaic virus family) andbromoviruses (brome mosaic virus family) and variants, derivatives ormodifications thereof Viral RNA replication can be regulated by themethods discussed below.

[0009] Therefore, it is one object of the present invention to provide amethod for controlling replication of viral RNA wherein cells areengineered to contain nucleic acid molecules (for example incorporatedinto the genome or into one or more vectors within the cells) having oneor more of the virus (e.g. alphavirus) non-structural protein genesunder the control of an inducible promoter. An expression construct(which may be RNA or DNA) containing the gene of interest under thecontrol of a promoter (preferably an alphavirus recognized promoter) andhaving a replication signal (preferably an alphavirus recognizedreplication signal) may be stably introduced into the cell (e.g. by wellknown transfection or transformation techniques) or introduced after thecells are grown to high mass. When using a DNA molecule, the expressionconstruct (containing the gene of interest under control of a promoterand the replication signal) is preferably under control of one or morepromoters (which may be inducible and/or constitutive). When the desiredRNA or protein is needed, the replicase or nonstructural genes areexpressed by activating the inducible promoter. The replicase in turnrecognizes the promoter and activates replication of the expressionconstruct and expression of the gene of interest, resulting in theproduction of the desired RNA or protein which can be harvested bystandard methods. It is another object of the present invention toprovide expression constructs and cells for use in the method describedabove for controlling expression of replicase and viral replication, andprotein production.

[0010] It is another object of the present invention to provide a methodfor controlling the amount of replicase (preferably alphavirusreplicase) and hence the amount of viral replication in a cell and tomethods of controlling expression of a desired protein using thissystem. Such methods may comprise introducing or transfecting cells,transiently or stably, with one or more replicase proteins (ornonstructural proteins) and/or with one or more replicase genes underregulateable control. The desired gene(s) to be expressed may becontained in one or more vectors may be contained in the genome of thecell. The gene of interest is preferably under the control of analphavirus recognized promoter such as the 26S promoter. Thus,replication and expression of the desired gene would not take place ornot be produced at elevated levels until one or more replicase ornonstructural proteins and/or other nonstructural genes are introduced,and gene expression and cell viability can thus be controlled untilappropriate cell growth has been achieved. Methods for the delivery ofone or more replicase or nonstructural proteins (e.g. nsp 1-4 proteins)are described. Expression constructs and cells for use in a method forcontrolling viral replication in a cell by introducing the nonstructuralproteins and/or genes (or combinations thereof) into a cell containing agene encoding a desired protein are also described.

[0011] It is yet another object of the present invention to provideexpression constructs and cells for use in a method for controllingviral replication and expression in a cell by introducing into the cella factor or drug which inhibits viral RNA replication.

[0012] As will be understood, these methods, expression constructs andcells may be applied to any number of RNA viral systems by usingappropriate replicase or nonstructural proteins/genes and geneticconstructs.

[0013] Other preferred embodiments of the present invention will beapparent to one of ordinary skill in the art in view of the followingdrawings and description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a schematic representation of possible arrangements ofthe nonstructural proteins, the promoters, and 5′ and 3′ sequences.Promoter =inducible or constitutive promoter; nsp 1-4, SFV nonstructuralproteins 1-4; 3′ SFV, SFV termination signals; A_(n), a stretch of >25nucleotides of Adenylic acid; Poly A signal, polyadenylation signal; nsp4*, only nonstructural protein 4 is used and will contain properlyspaced start and stop codons for proper expression of the protein; nsp1+2+4, nonstructural protein 1, 2, and 4 are included; 26S promoter,subgenomic promoter recognized by the SFV replicase; 5′SFV, SFVreplication signal; MCS, multiple cloning site containing endonucleaserestriction sites for ease of inserting the gene of interest.

[0015]FIG. 2 shows the possible arrangements of a construct with thegene of interest. Promoter: inducible or constitutive promoter; 5′ SFV,SFV replication signal; 26 S promoter, subgenomic promoter recognized bySFV replicase; gene, gene of interest; internal ribosome entry sites(IRES), antibiotic gene, antibiotic resistance gene, or a selectablemarker for selecting transformants.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In the description that follows, a number of terms used inrecombinant DNA technology are extensively utilized. In order to providea clearer and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

[0017] Replicon. Any genetic element, e.g. a plasmid, a chromosome, avirus, that behaves as an autonomous unit of polynucleotide replicationwithin a cell; i.e., capable of replication under its own control.

[0018] Cloning vector. A plasmid, cosmid or phage or other nucleic acidmolecule (e.g. RNA or DNA) which is able to replicate autonomously in ahost cell, and which is characterized by one or a small number ofrestriction endonuclease recognition sites at which such sequences maybe cut in a determinable fashion without loss of an essential biologicalfunction of the vector, and into which nucleic acid may be spliced inorder to bring about its replication and cloning. The cloning vector mayfurther contain a marker suitable for use in the identification of cellstransformed with the cloning vector.

[0019] Expression vector. A vector similar to a cloning vector but whichis capable of enhancing the expression of a gene which has been clonedinto it, after transformation or transfection into a host. The clonedgene is usually placed under the control of (i.e., operably linked to)certain control sequences such as promoter sequences.

[0020] Recombinant host. Any prokaryotic or eukaryotic organism,microorganism or bacteria which contains the vector or vectors, orcontains the desired cloned genes in an expression vector, cloningvector of any nucleic acid molecule. The term “recombinant host” is alsomeant to include those host cells which have been genetically engineeredto contain the desired vectors or genes on the host chromosome orgenome.

[0021] Host. Any prokaryotic or eukaryotic organism, microorganism orbacteria that is the recipient of a replicable expression vector,cloning vector or any nucleic acid molecule. The molecule may contain,but is not limited to, a structural gene, a promoter and/or an origin ofreplication.

[0022] Gene. A nucleic acid sequence that contains information necessaryfor expression of a polypeptide or protein. It includes the promoter andthe structural gene as well as other sequences involved in expression ofthe protein. When a gene is under the control of a 26S promoter, therewill generally be some or no 5′ untranslated region, an ATG at the startof the coding sequence and a stop codon. The gene of interest may alsocomprise additional sequences to create fusion proteins to facilitateprotein purification, such as His tags, GST tags and the like.

[0023] Expression construct. Any type of genetic construct containing anucleic acid coding for a gene product in which part or all of thenucleic acid encoding sequence is capable of being transcribed. Thetranscript may be translated into a protein, but it need not be. Thus,in certain embodiments, expression includes both transcription andtranslation of a gene into a gene product. In other embodiments,expression only includes transcription of the nucleic acid encoding thegene, which can be useful for making RNA for antisense gene therapy

[0024] Promoter. A nucleic acid sequence or transcription controlelement generally described as the 5′ region of a gene and/or othersequence, located proximal to the start codon. At the promoter region,transcription of an adjacent gene(s) or sequence(s) is initiated.

[0025] Constitutive promoter. A transcription control element which iscontinuously functional, i.e., cannot be induced nor repressed, andresults in the continuous expression of a gene operably linked to it.Constitutive promoters include promoters for housekeeping genes, orgenes which are continuously expressed in the cell. Examples of suchpromoters include human cytomegalovirus (CMV) immediate early genepromoter, the SV40 early promoter and the Rous sarcoma virus longterminal repeat.

[0026] Inducible promoter. A transcriptional control element which canbe regulated in response to specific signals. Table 1 lists severalpromoters which may be employed in the context of the present inventionto regulate the expression of genes or sequences operably linked to suchpromoters. TABLE 1 Promoters and Their Inducible Factors Element Inducer 1. MTII Phorbol Ester (TPA), Heavy metals  2. MMTV (mouse mammaryGlucocorticoids    tumor virus)  3. b-interferon poly(rI)X, poly(rc)  4.Adenovirus 5 E2 Ela  5. c-jun Phorbol Ester(TPA), H₂O₂  6. CollagenasePhorbol Ester(TPA)  7. Stromelysin Phorbol Ester(TPA), IL-1  8. SV-40Phorbol Ester(TPA)  9. Murine MX Gene Interferon, Newcastle DiseaseVirus 10. GRP78 Gene A23187 11. a-2-macroglobulin IL-6 12. VimentinSerum 13. HSP70 Ela, SV-40 Large T Antigen 14. Tumor Necrosis Factor FMA15. Thyroid Stimulating Hormone Thyroid Hormone 16. Tetracyclineregulated Promoter Tetracycline 17. Ecdysone inducible PromoterEcdysone, Muristerone, Ponasterone A

[0027] Bicistronic vector. A nucleic acid vector with an arrangement ofpromoter and two sequences resulting in RNA transcripts produced fromthe same start site, one encompassing a first sequence, and anothertranscript including the first sequence and the sequence 3′ of the firstsequence.

[0028] Polyadenylation signal. A nucleic acid sequence which allows theaddition of a sequence of polyadenylic acid to the 3′ end of a RNA(preferably eukaryotic RNA) after its transcription. The nature orpresence of the polyadenylation signal is not believed to be crucial tothe successful practice of the invention, and any such sequence may beemployed if its use is desired. The SV40 polyadenylation signal was usedfor convenience and known to function in the target cells of interest.Also contemplated as an element of an introduced nucleic acid is atermination signal which serves to minimize read through.

[0029] Marker. A nucleic acid sequence coding for a marker, usuallycloned into a vector for use in the identification of cell transformedor transfected with the vector. A marker would result in an identifiablechange to the transfected or transformed cell permitting easyidentification of expression or delivery of nucleic acid in vitro or invivo. Usually the inclusion of a drug selection marker aids in cloningand in the selection of transformants or transfectants. Alternatively,enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic)or chloramphenicol acetyltransferase (CAT) (prokaryotic) may beemployed. Immunologic markers also can be employed. Further examples ofselectable markers are known to one of skill in the art.

[0030] Delivery of nucleic acid to cells. Delivery of the desirednucleic acids to cells or host cells can be accomplished in vitro, as inlaboratory procedures for transforming or transfecting cell lines, or invivo, or ex vivo. Methods include calcium phosphate precipitation(Graham and Ven Der Eb (1973); Chen and Okayama, (1987); Rippe etal.(1990)), DEAE Dextran (Gopal, (1985)), electroporation (Tur-Kaspa etal. (1986); Porter et al., (1984)), direct microinjection (Harland andWeintraub, (1985)), DNA-loaded liposomes (Nicolau and Sene, (1982);Fraley et al. (1979)) and lipofectin-DNA or cationic lipid-DNA(Hawley-Nelson, et al., Lipofectamine (1993) Focus, 15: 73-78;Ciccarone, et al., DMRIE-C (1995) Focus, 17: 84-87) complexes, cellsonication (Felgner et al., (1987)), gene bombardment using highvelocity microprojectiles (Yang et al., (1990)) and receptor-mediatedtransfection (Wu and Wu, (1987); Wu and Wu, (1988)). Some of thesetechniques may be successfully adapted for in vivo or ex vivo use. Howthe nucleic acid is delivered to a cell is dependent on the type ofnucleic acid employed.

[0031] Stable cell line. Once the nucleic acid (e.g. RNA and/or DNA) hasbeen delivered into the cell, the nucleic acid may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidmay be stably integrated into the genome of the cell, producing a stablecell line. This integration may be in the cognate location andorientation via homologous recombination, or it may be integrated in arandom non-specific location. The nucleic acid may be stably maintainedin the cell as a separate, episomal segment. Such nucleic acid segmentsor “episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle, wherein the cell the nucleic acid remains is dependent on thetype of vector employed.

[0032] The present invention describes methods, vectors, cell lines, andagents for the regulated control of gene expression using an alphavirusreplicon or other RNA viral systems.

[0033] In one embodiment, the present invention provides a method forthe regulated control of alphavirus or other virus replicon and geneexpression by controlling the amount of replicases or nonstructuralproteins expressed by a cell line. As such, using the expression systemof the present invention, one can produce a cell line containing thenucleic acid molecules capable of expressing one or more genes encodingnonstructural proteins (e.g. nsp 1-4), or some combination of thesenonstructural proteins, under the control of an inducible and/orconstitutive promoter. Preferably, at least one nonstructural proteingene is under control of an inducible promoter. This can be done, forexample, by introducing a vector or vectors containing one or more ofthe nonstructural protein genes operably linked to a promoter (differentcombinations of constitutive and inducible promoters may be used butpreferably at least one replicase or nonstructural protein gene is undercontrol of an inducible promoter) into the desired cells. The vector canbe any vector but is preferably a DNA vector. The one or morenonstructural protein genes may be integrated into the host genome ormay be contained in one or more vectors or various combinations of suchgenes may be both integrated in the genome and contained in one or morevectors. For integration, a constitutive promoter controlling expressionof a selectable marker (neo, hygro, puro, blasticidin) is preferred. Forepisomal or vector maintenance, a selectable marker (as above) and anorigin of replication (EBNA, etc.) is required. The vector alsopreferably contains an E. Coli origin of replication and a selectablemarker with a prokaryotic promoter for selection and maintenance in E.coli, although other prokaryotic or eukaryotic origins and/or markersmay be used depending on the need or host cell used. In another aspectof the invention, the gene of interest or expression construct may becontained by the same vector/genome having the one or more nonstructuralprotein genes.

[0034] The cells containing the one or more genes for the nonstructuralproteins are allowed to multiply until the desired number of cells orcell mass is reached. The gene of interest to be expressed under thecontrol of one or more transcriptional control elements or promoterspreferably recognized by the nonstructural viral replicases, such as the26S promoter for SFV, can be induced when the desired number of cells orcell mass has been reached thereby producing high levels of the desiredprotein. Preferably, the genes of interest are contained by one or moreexpression vectors which may be integrated into the host genome ormaintained as one or more expression replicons (or combinationsthereof). Following induction of replication, and gene expression, cellscan be harvested to isolate the protein of interest.

[0035] For example, genes encoding some or all of the nonstructuralproteins (or various combinations thereof) can be positioned under aconstitutive or a regulated promoter. Preferably, at least onenonstructural protein gene is under control of a regulatable promoter.When nsp 4 is the only nonstructural protein gene used, the nsp 4 willcontain a properly spaced translation initiation codon (ATG) and atranslation termination codon (TAA). The promoter sequences used in theinvention may be constitutive promoters such as cytomegalovirus (CMV)promoter (Boshart, M. et al. (1985) Cell 41:521-530) or an induciblepromoter system such as the tetracycline-regulated promoter (Gossen, M.and Bujard, H. (1992) PNAS USA 89:5547-5551; Shockett, P. et al. (1995)PNAS USA 92:6522-6526) or the ecdysone inducible promoter (No, D. etal.(1996) PNAS USA 93:3346-3351). The nsp 1, 2, 3, and 4 are thenon-structural proteins of SFV and the 5′ SFV signal contains thesequences necessary for replication (Levis R. et al. (1990) J. Virol.64:1726; Grakoui, A. et al. (1989) J. Virol. 63:5216). The 3′ end of theconstruct will include 3′ SFV termination signals (Kuhn, R. J. et al.(1990) J. Virol. 64:1465), a stretch of 25 nucleotides of A, and theSV40 termination and polyadenylation signals.

[0036] The nonstructural proteins, the inducible promoter and the 5′ and3′ sequences can be arranged in several ways as diagramed in FIG. 1.

[0037] Stable cell lines are generated with constructs 1-3 of FIG. 1that express all or some of the SFV nonstructural proteins under controlof either a constitutive promoter (i.e. CMV) or an inducible promoter(tetracycline-inducible minimal CMV, or ecdysone inducible-pIND). Forecdysone or muristerone inducible expression, the vectors areco-transfected with a vector encoding the ecdysone responsive elementpVgRXR (No et al., 1996, supra). Stable transfection of any mammalian orinsect cell line (e.g. CHO, BHK, Drosophila, Spodoptera cell lines) isachieved by either co-transfection with a second vector encoding apositive selectable marker (neo^(r), blasticidin^(r), hygro^(r),puromycin^(r)) or by incorporating the selectable marker on the samevector. The selected clonal cell lines will then express the one or moreof the nonstructural genes either constitutively (for CMV promoter) orupon induction by either a) removal of tetracycline from medium, or b)addition of the ecdysone analog, muristerone or ponasterone A to thecell medium.

[0038] These established cell lines can then be transiently or stablytransfected with nucleic acid molecules (e.g. RNA or DNA, preferablyvectors) encoding a gene of interest expressed under control of, forexample, the SFV 26S promoter, and are thus capable of generatingreplicating RNA molecules using the nonstructural proteins of, forexample, SFV or other alphaviruses. RNA replication is dependent on theSFV nonstructural protein 4 replicase, and may be achieved by thepresence of the 5′ SFV replication signal (as in construct 4 of FIG. 1).Thus, replication and expression of the SFV-encoded RNA would not takeplace or would not be produced at elevated levels until the cells areinduced to express the replicase and/or other SFV nonstructural genes,and gene expression and cell viability can thus be controlled untilappropriate cell growth has been achieved. Such control will allowmaximum production of the protein of interest. In the case where thestable cell line encodes the nonstructural genes under a constitutivepromoter, the expression construct or vector or any expression vector orreplicon or nucleic acid molecule of interest containing the gene to beexpressed is transiently transfected or transformed into large scalecultures and replication and expression begin directly aftertransfection or transformation. Where inducible regulation is used, suchexpression vectors or replicons may be introduced into the cell line atany time and protein expression may then be accomplished upon inductionof expression of the one or more nonstructural proteins. Cells areharvested for protein isolation (typically 24-48 hourspost-transfection).

[0039] This strategy can also be achieved by stable transfection of asingle vector or construct, as in constructs 5-7 of FIG. 1, where boththe gene of interest and one or more nonstructural genes are encoded onthe same vector, and expression of the nonstructural genes is regulatedby an inducible promoter. Stable cell lines that contain such vectors orconstructs are scaled up to appropriate cell mass and induced to expressnonstructural proteins, thus allowing expression of the gene of interestfrom for example the 26S promoter and replication of RNA.

[0040] In another embodiment, vectors encoding a gene of interest andantibiotic resistance gene (for example, puromycin^(r), hygromycin^(r)or blasticidin^(r) for rapid selection), under control of SFV 26Spromoter (as in construct 8 of FIG. 2), is transfected into a cell linethat expresses the SFV nsp genes under control of an inducible promoter.The cells are placed under selective pressure by addition of theantibiotic, then grown to a high cell mass. At this point the cells areinduced to express the nsp genes, which in turn drive RNA replicationand overexpression.

[0041] Alternatively, a bicistronic vector can be created (as in vector9 of FIG. 2) which expresses both the antibiotic resistance gene andgene of interest under control of a single 26S promoter. Thus any cellthat expresses antibiotic resistance must also express the gene ofinterest. Similarly, this vector is transfected into a stable cell linewhich expresses the SFV nsp genes from an inducible promoter. Afterantibiotic selection, the promoter that drives expression of thereplicase is induced, and cells are harvested for the protein ofinterest.

[0042] In yet another embodiment of the present invention is a methodfor controlling alphavirus or other virus replicon expression byinitiating replication and protein expression in cells by introducingone or more nonstructural proteins (preferably one or more SFVnonstructural proteins) into the cells that contain the gene of interestunder control of one or more promoters (e.g. 26S promoters). As will beappreciated, the amount of protein introduced may be used to regulatethe level of expression of the gene of interest (i.e. introduction ofmore nonstructural proteins may be used to increase expression) and theamount of protein introduced can be optimized for the particular systemused. The nonstructural proteins in any combination may be introducedinto cells by methods known to a person with skill in the art. Forexample, proteins can be delivered to a cell using cationic lipids, suchas lipofectamine or DMRIE-C (Sells, et al, (1995) Biotechniques, 19:72-75). Alternatively, a fusion protein with a peptide or proteinsequence which has a transport function can be used. For example, theHIV Tat protein or a peptide component of Tat (Mann and Frankel (1991)EMBO J. 10:1733-1739; Frankel et al. (1989) PNAS USA 86:7397-7401) andthe Herpesvirus VP22 protein (Elliot, G. et al. (1997) Cell 88:223-233)and membrane translating sequence (MTS) (Rojas, et al., (1998) NatureBiotechnology, 16: 370-375) have been shown to mediate the uptake intocells of a heterologous protein when the protein is synthesized as afusion with the transport protein or peptide. As will be recognized,some nonstructural proteins may be introduced by these introductionmethods while other proteins may be expressed by these introductionmethods described above. Thus, various combinations of nonstructuralprotein expression and introduction of nonstructural proteins may beutilized in accordance with the invention.

[0043] For example, a stable cell line is constructed which contains theSFV nsp 1-3 genes under an inducible and/or constitutive promoters (ifunder the CMV promoter) (as in vectors 1-3 of FIG. 1) and, on the sameor separate vector, the gene of interest is under control of the SFV 26Spromoter (as in vectors 10 and 11 of FIG. 2). Expression of the nsp 1-3genes is therefore constitutive or induced to drive expression. Once thedesired cell population density is achieved, RNA replication andenhanced gene expression is achieved by introduction of the nsp 4replicase protein into the cells. The protein can be introduced withcationic lipids or, more efficiently, as synthetic fusions with apeptide or protein that has a transport function and allows the fusedprotein to cross the cell and nuclear membranes (i.e. as Tat, MTS orVP22 fusion proteins).

[0044] Therefore, gene expression can be induced at moderate to highlevels in the cell population, while retaining viability, by expressionof the nsp 1-3 genes and subsequently activation of the 26S promoter.Once cell confluency is reached, replication can be induced by deliveryof the nsp 4 replicase protein (at various amounts or concentrations)and cells can then be harvested for protein isolation (e.g. hourslater).

[0045] Alternatively, the nsp 1-4 proteins can also be delivered tocells. In this case, cells are stably transfected with a vector thatencodes the gene of interest under control of the SFV 26S promoter (asin vector 10, 11, or 12 of FIG. 2). Both gene expression and RNAreplication are induced by delivery of nsp 1-4 proteins as a complexwith cationic lipids or as fusion proteins.

[0046] In yet another embodiment of the present invention, a drug oragent which inhibits viral replication is introduced into a cell.Alphavirus or other virus RNA replication can be inhibited by proteinsor drugs which have antiviral activity. Mx proteins, for example, whichare GTPases induced by interferons (Staehli, P. (1990) Adv. Virus Res.38:147-200) have been shown to inhibit the multiplication ofnegative-strand RNA viruses (Frese, M. et al.(1996) J. Virol.70:915-923; Meier, E. et al.(1990) J. Virol. 64:6263-6269; Pavolvic, J.et al. (1990) J. Virol. 64: 3370-3375; Zhao, H. et al.(1996) Virology220:330-338). The human MxA protein, specifically, was found to blockthe multiplication of influenza virus at the level of RNA synthesis orsynthesis of viral glycoproteins (Schneider-Schaulies, S. et al.(1994)J. Virol. 68:6910-6917; Schnorr, J. et al.(1993) J. Virol.67:4760-4768), and was also found to have antiviral activity to SFV byinhibiting RNA replication at the level of synthesis or function of theviral replicase (Landis, H. et al.(1998) J. Virol. 72: 1516-1522). TheMxA protein can therefore be utilized to control replication ofrecombinant RNA virus (e.g. SFV) and therefore regulate gene expression.

[0047] For example, a stable cell line is constructed with human MxAprotein under control of an inducible promoter (e.g. a tetracycline ormuristerone-regulated promoter). The expression of the MxA protein iskept in the induced state until RNA replication is required. This stablecell line is then transfected with a vector such as construct 5 of FIG.1 or a combination of constructs (e.g. construct 1 (FIG. 1) andconstruct 10 or 11 (FIG. 2)) to express the nsp genes and the gene ofinterest. RNA replication, however, will be inhibited or prevented bythe expression of the MxA protein. When MxA induced expression is shutoff either by addition of tetracycline or removal of muristerone fromthe medium, RNA replication and expression is allowed.

[0048] When the desired protein is expressed and produced by thetransfected or transformed cell to the desired level, the protein can becollected either from the supernatant, if the protein is secreted, or byharvesting the cells and extracting the desired protein by methods knownin the art, and specific for the desired protein.

[0049] The methods, cells and vectors of the present invention can beused to produce therapeutic compounds and/or to provide therapeuticcompounds to specific cells, tissues, organs or organisms. Therapeuticcompounds include protein, RNA, and DNA cloned into the vectors forproviding a clinical effect. Proteins provided for therapeutic purposeswould include antigens for the production of an immunogenic responseagainst a pathogen, or a drug for treatment of a disease or diseasesymptoms. RNA could be provided for the production of a protein asdescribed above, or alternatively, antisense RNA is provided such thattranslation of an undesired protein or factor is inhibited or reduced.DNA can be provided encoding a protein or RNA as described above, andfurther for a marker, or for expression of a gene operably linked tosuch DNA allowing a drug to effect the expression of a desired gene, orfor integration into the host genome in order to disrupt a targetedgene.

[0050] Where clinical applications of the present invention arecontemplated, it may be necessary to prepare the complex as apharmaceutical composition appropriate for the intended application.Generally this will entail preparing a pharmaceutical composition thatis essentially free of pyrogens, as well as any other impurities thatcould be harmful to humans or animals. One also will generally desire toemploy appropriate salts and buffers to render the complex stable andallow for complex uptake by target cells.

[0051] Aqueous compositions of the present invention comprise aneffective amount of the expression construct and/or cells of theinvention, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions can also be referred to asinocula. The phrases “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, or a human, as appropriate. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic, and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients also can be incorporatedinto the compositions.

[0052] Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hyroxypropylcellulose. Dispersions alsocan be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0053] The expression constructs and delivery vehicles of the presentinvention may include classic pharmaceutical preparations.Administration of therapeutic compositions according to the presentinvention will be via any common route so long as the target tissue orcell is available via that route. This includes oral, nasal, buccal,rectal, vaginal, or topical. Topical administration would beparticularly advantageous for treatment of skin cancers, for example.Alternatively, administration will be by orthotopic, intradermalsubcutaneous, intramuscular, intraperitoneal or intravenous injection.Such compositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers, or other excipients.

[0054] The therapeutic compositions of the present invention areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified. A typicalcomposition for such purpose comprises a pharmaceutically acceptablecarrier. For instance, the composition may contain 10 mg, 25 mg, 50 mgor up to about 100 mg of human serum albumin per milliliter of phosphatebuffered saline. Other pharmaceutically acceptable carriers includeaqueous solutions, non-toxic excipients, including salts, preservatives,buffers and the like. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oil and injectable organic esterssuch as ethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactcomposition are adjusted according to well known parameters.

[0055] Additional formulations are suitable for oral administration.Oral formulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray. An effectiveamount of the therapeutic agent is determined based on the intendedgoal. The term “unit dose” refers to physically discrete units suitablefor use in a subject, each unit containing a predetermined quantity ofthe therapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. Precise amounts of the therapeutic compositions alsodepend on the judgement of the practitioner and are peculiar to eachindividual.

[0056] All the materials and reagents required for expressing a desiredgene using the present invention may be assembled together in a kit.This generally will comprise one or more of the following: selected celllines which are capable of expressing or accepting the viralnonstructural proteins, and/or constructs to be used along with the cellline in which a gene or genes of interest can be inserted such that theymay be expressed when introduced into the cell line. Also included maybe various media or antibiotics for replication and selection of hostcells, as well as drugs or agents suitable for inhibiting viralreplication. Additionally, the kit may include transfection reagents,i.e. cationic lipid transfection reagents. Such kits may comprisedistinct containers for each individual reagent or such reagents may becombined in containers in various combinations and concentrations.

[0057] When the components of the kit are provided in one or more liquidsolutions, the liquid solution preferably is an aqueous solution, with asterile aqueous solution being particularly preferred.

[0058] The components of the kit may also be provided in dried orlyophilized forms. When reagents or components are provided as a driedfrom, reconstitution generally is by the addition of a suitable solvent.It is envisioned that the solvent also may be provided in anothercontainer means.

[0059] The kits of the present invention also will typically include ameans for containing the vials in close confinement for commercial salesuch as, e.g. injection or blow-molded plastic containers into which thedesired vials are retained.

[0060] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples representtechniques discovered by the inventors and thought to function well inthe practice of the invention, and thus can be considered to constitutepreferred, but non-limiting, modes for its practice. However, those ofskill in the art should, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments which aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope of the invention.

EXAMPLES Example 1 Cloning of Non-Structural Proteins Under aConstitutive or a Regulated Promoter

[0061] The promoter sequence is either a constitutive promoter such ascytomegalovirus (CMV) promoter (Boshart, M. et al. (1985) Cell41:521-530) or an inducible promoter system such as thetetracycline-regulated promoter (Gossen, M. and Bujard, H. (1992) PNASUSA 89:5547-5551; Shockett, P. et al. (1995) PNAS USA 92:6522-6526) orthe ecdysone inducible promoter (No, D. et al. (1996) PNAS USA93:3346-3351). The nsp 1,2,3, and 4 are the non-structural proteins ofSFV and the 5′ SFV signal contains the sequences necessary forreplication (Levis R. et al. (1990) J. Virol. 64:1726; Grakoui, A. etal.(1989) J. Virol. 63:5216). The 3′ end of the construct will include3′ SFV termination signals (Kuhn, R. J. et al. (1990) J. Virol.64:1465), a stretch of 25 nucleotides of A, and the SV40 termination andpolyadenylation signals.

[0062] The nonstructural proteins, the inducible promoter and thenecessary 5′ and 3′ sequences can be arranged in several ways asdiagramed in FIG. 1.

[0063] Stable cell lines are generated with constructs 1-3 of FIG. 1that express all or some of the SFV nonstructural proteins under controlof either a constitutive promoter (e.g., CMV) or an inducible promoter(tetracycline-inducible minimal CMV, or ecdysone inducible-pIND). Forecdysone, ponasterone A or muristerone inducible expression, the vectorsare co-transfected with a vector encoding the ecdysone responsiveelement pVgRXR (No et al., 1996, supra). Stable transfection of anymammalian or insect cell line (e.g. CHO, BHK, Drosophila, Spodopteracell lines) is achieved by either co-transfection with a second vectorencoding a positive selectable marker (neo^(r), blasticidin^(r),hygro^(r), puromycin^(r)) or by incorporating the selectable marker onthe same vector. The selected clonal cell lines will then express theSFV nonstructural genes and protein either constitutively (for CMVpromoter) or upon induction by either a) removal of tetracycline frommedium, or b) addition of the ecdysone analog, muristerone orponasterone A to the cell medium.

[0064] These established cell lines can then be transiently or stablytransfected with vectors encoding a gene of interest expressed undercontrol of the SFV 26S promoter, and are thus capable of generatingreplicating RNA molecules using the nonstructural proteins of SFV orother alphavirus. RNA replication is dependent on the SFV nonstructuralprotein 4 replicase, and may be achieved by the presence of the 5′ SFVreplication signal (as in construct 4 of FIG. 1). Thus, replication andexpression of the SFV-encoded RNA would not take place at high levelsuntil the cells are induced to express the replicase and other SFVnonstructural genes, and gene expression and cell viability can thus becontrolled until appropriate cell growth has been achieved. In the casewhere the stable cell line encodes the nonstructural genes under aconstitutive promoter, the SFV expression vector is transientlytransfected into large scale cultures and replication and expressionbegins directly after transfection. Cells are harvested for proteinisolation 24-48 hours post-transfection. Alternatively, these stablecell lines can also be transfected with a truncated SFV replicon withoutthe nsp such that the RNA molecule will not replicate until the stablecell is induced to replicate.

[0065] This strategy can also be achieved by stable transfection of asingle vector, as in constructs 5-7 of FIG. 1, where both the gene ofinterest and nonstructural genes are encoded on the same vector, andexpression of the nonstructural genes is regulated by an induciblepromoter. Stable cell lines that contain this vector are scaled up toappropriate cell mass and induced to express nonstructural proteins,thus allowing expression of the gene of interest from the 26S promoterand replication of RNA.

Example 2 Expression Constructs with Selectable Marker Under 26sPromoter and Bicistronic Vectors for Use with Stable Cell Line with nspGenes

[0066] Vectors encoding gene of interest and antibiotic resistance gene(blasticidinr for rapid selection), under control of SFV 26S promoter(as in construct 8 of FIG. 2), are transfected into a cell line thatexpresses the SFV nsp genes under control of an inducible promoter. Thecells are placed under selective pressure by addition of the antibiotic,then grown to a high cell mass. At this point the cells are induced toexpress the nsp genes, which in turn drive RNA replication andoverexpression.

[0067] Alternatively, a bicistronic vector can be created (as in vector9 of FIG. 2) which expresses both the antibiotic resistance gene andgene of interest under control of a single 26S promoter. Thus any cellthat expresses antibiotic resistance must also express the gene ofinterest. Similarly, this vector is transfected into a stable cell linewhich expresses the SFV nsp genes from an inducible promoter. Afterantibiotic selection, the promoter that drives expression of thereplicase is induced, and cells are harvested for protein.

Example 3 Induction of SFV RNA Replication by Introduction of ReplicaseProtein into Cells

[0068] A stable cell line is constructed which contains the SFV nsp 1-3genes under an inducible or constitutive promoter (if under the CMVpromoter) (as in vectors 1-3 of FIG. 1) and, on the same or separatevector, the gene of interest is under control of the SFV 26S promoter(as in vectors 10 and 11 of FIG. 2). Expression of the nsp 1-3 genes istherefore constitutive or induced to drive expression of the genes ofinterest. Once the desired cell population density is achieved, RNAreplication and enhanced gene expression is achieved by introduction ofthe nsp 4 replicase protein into the cells. The protein can beintroduced with cationic lipids or, more efficiently, as syntheticfusions with a peptide or protein that has a transport function andallows the fused protein to cross the cell and nuclear membranes (i.e.as Tat, MTS or VP22 fusion proteins).

[0069] Therefore, gene expression can be induced at moderate to highlevels in the cell population, while retaining viability, by expressionof the nsp 1-3 genes and subsequently activation of the 26S promoter.Once cell confluency is reached, replication can be induced by deliveryof the nsp 4 replicase protein and cells can be harvested for proteinisolation hours later.

[0070] Alternatively, the nsp 1-4 proteins can also be delivered tocells. In this case, cells are stably transfected with a vector thatencodes the gene of interest under control of the SFV 26S promoter (asin vector 10, 11, or 12 of FIG. 2). Both gene expression and RNAreplication are induced by delivery of nsp 1-4 proteins as a complexwith cationic lipids or as fusion proteins.

Example 4 Regulation of RNA Replication with MxA Protein

[0071] A stable cell line is constructed which expresses the human MxAprotein under control of a tetracycline or muristerone-regulatedpromoter. The expression of the MxA protein is kept in the induced stateuntil RNA replication is required. This stable cell line is thentransfected with a vector such as construct 5 of FIG. 1 or a combinationof construct 1 (FIG. 1) and construct 10 or 11 (FIG. 2) to express thensp genes and the gene of interest. RNA replication, however, will beinhibited by the expression of the MxA protein. When MxA expression isshut off either by addition of tetracycline or removal of muristeronefrom the medium, RNA replication and expression is allowed.

[0072] The experimental designs described above are therefore aimed todesign a regulated alphavirus expression system, where the geneexpression is controlled by controlling the replication of the RNAreplicon. The benefit of such a system is to take advantage of the highlevel expression of the alphavirus systems for recombinant proteinproduction for large scale applications without the biosafety concernsof utilizing a packaged virus.

[0073] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[0074] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference.

What is claimed is:
 1. A method for controlling viral RNA replication orgene expression in a cell, said method comprising controlling expressionof said one or more genes encoding one or more viral replicases ornonstructural proteins in said cell and/or introducing one or more ofsaid replicases or nonstructural proteins in said cell.
 2. The methodaccording to claim 1, wherein one or more of said genes is under controlof an inducible promoter.
 3. The method according to claim 1, whereinsaid method controls alphavirus RNA replication.
 4. The method accordingto claim 1, wherein said one or more genes is under control of aconstitutive promoter.
 5. The method according to claim 1, wherein saidmethod further comprises controlling expression of one or more genes ofinterest.
 6. The method according to claim 5, wherein one or more ofsaid genes of interest are under control of a viral promoter.
 7. Themethod according to claim 1, wherein one or more of said replicases ornonstructural proteins are introduced into said cell by using one ormore cationic lipids.
 8. The method according to claim 1, wherein saidone or more replicases or nonstructural proteins are introduced intosaid cell by fusing said replicases or proteins with a peptide orprotein sequence having a transport function such that said replicaseprotein is transported into said cell.
 9. The method according to claim3, wherein said alphavirus is selected from the group consisting ofSemliki Forest Virus, Sinbis Virus, Venezuelan Equine EncephalitisVirus, Eastern Equine Encephalitis Virus.
 10. The method according toclaim 1, wherein said replicases or nonstructural proteins are selectedfrom the group consisting of nonstructural proteins 1, 2, 3 and 4 andcombinations thereof.
 11. A nucleic acid molecule comprising one or moregenes encoding one or more viral replicases or nonstructural proteinsunder control of one or more inducible promoters.
 12. The nucleic acidmolecule according to claim 11, wherein said one or more genes encodefor nonstructural proteins 1, 2, 3 or 4 or combination thereof.
 13. Thenucleic acid molecule according to claim 11, wherein said viralreplicases or nonstructural proteins are derived from an alphavirus. 14.The nucleic acid molecule according to claim 13, wherein said alphavirusis selected from the group consisting of Semliki Forest Virus, SinbisVirus, Venezuelan Equine Encephalitis Virus and Eastern EquineEncephalitis Virus.
 15. The nucleic acid molecule according to claim 11,wherein said molecule is a vector.
 16. The nucleic acid moleculeaccording to claim 11, wherein one or more of said genes is undercontrol of one or more constitutive promoters.
 17. The nucleic acidmolecule according to claim 11, further comprising one or more genes ofinterest under control of a viral promoter.
 18. The nucleic acidmolecule according to claim 17, wherein said viral promoter is analphavirus recognized promoter.
 19. The nucleic acid molecule accordingto claim 18, wherein said alphavirus is selected from the groupconsisting of Semliki Forest Virus, Sinbis Virus, Venezuelan EquineEncephalitis Virus, Eastern Equine Encephalitis Virus.
 20. A host cellcomprising the nucleic acid molecule according to claim
 11. 21. A hostcell comprising the nucleic acid molecule according to claim
 17. 22. Apharmaceutical composition comprising a cell of claim 20 and apharmaceutical carrier.
 23. A pharmaceutical composition comprising acell of claim 21 and a pharmaceutical carrier.
 24. A method forcontrolling expression of a gene of interest in a cell comprisingproviding a factor or drug which inhibits or prevents viral RNAreplication in said cell.
 25. The method of claim 24, wherein saidfactor is MxA.